The objective of this proposal is to address the pathogenesis of gastrointestinal disease associated with Shiga-like toxin (SLT) producing E. coli, which colonize the gastrointestinal tract. SLTs are considered to be responsible for both local and systemic disease. This proposal will focus on the interactions of the SLTs with intestinal epithelial cells (IECs) and endothelial cells, address how SLTs cross IECs, what effect SLTs have on IECs and how the combination of cytokines, SLTs and lipopolysaccharides (LPS) contribute to endothelial cell damage resulting in thrombotic microangiopathy (TMA) in the gut. Three specific aims are proposed. The first will determine how SLTs cross IECs using in vitro models. The effect of SLTs on the production of cytokines and the role of the bacteria in these interactions. The second specific aim is directed toward understanding how SLTs interact with, and cross IECs in vivo from bacterial infection within the gut. The third aim will study the effect of SLTs, cytokines from IECs and LPS on endothelial cell markers important in TMA. The methods to be used involve in vitro experiments using IECs in tissue culture and intestinal tissue in Ussing chambers to better understand SLT movement. Cytokine expression will be determined using RT-PCR and immunoassays. The in vivo experiments will be undertaken using SLT-producing bacteria and isogenic controls known to colonize either mice or rabbits to study both toxin interaction and cytokine production. Endothelial cell experiments will involve both tissue culture and in vivo studies and determination of TMA-associated factors in response to SLTs, cytokines and LPS. Shiga-like toxins produced in the gastrointestinal tract are associated with bloody and non-bloody diarrhea, as well as systemic disease (hemolytic uremic syndrome). It is anticipated that by gaining an understanding of SLT translocation and interaction with endothelial cells, considered to be the main target of this disease, it will ultimately allow the development of directed therapy to prevent SLT translocation and damage in the intestinal tract, and thereby reduce the clinical severity of the infection with SLT-producing E. coli.